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1.
Mol Med Rep ; 30(3)2024 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-38963029

RESUMO

Viral infections in the respiratory tract are common, and, in recent years, severe acute respiratory syndrome coronavirus 2 outbreaks have highlighted the effect of viral infections on antiviral innate immune and inflammatory reactions. Specific treatments for numerous viral respiratory infections have not yet been established and they are mainly treated symptomatically. Therefore, understanding the details of the innate immune system underlying the airway epithelium is crucial for the development of new therapies. The present study aimed to investigate the function and expression of interferon (IFN)­stimulated gene (ISG)60 in non­cancerous bronchial epithelial BEAS­2B cells exposed to a Toll­like receptor 3 agonist. BEAS­2B cells were treated with a synthetic TLR3 ligand, polyinosinic­polycytidylic acid (poly IC). The mRNA and protein expression levels of ISG60 were analyzed using reverse transcription­quantitative PCR and western blotting, respectively. The levels of C­X­C motif chemokine ligand 10 (CXCL10) were examined using an enzyme­linked immunosorbent assay, and the effects of knockdown of IFN­ß, ISG60 and ISG56 were examined using specific small interfering RNAs. Notably, ISG60 expression was increased in proportion to poly IC concentration, and recombinant human IFN­ß also induced ISG60 expression. By contrast, knockdown of IFN­ß and ISG56 decreased ISG60 expression, and ISG60 knockdown reduced CXCL10 and ISG56 expression. These findings suggested that ISG60 is partly implicated in CXCL10 expression and that ISG60 may serve a role in the innate immune response of bronchial epithelial cells. The present study highlights ISG60 as a potential target for new therapeutic strategies against viral infections in the airway.


Assuntos
Brônquios , Quimiocina CXCL10 , Células Epiteliais , Poli I-C , Transdução de Sinais , Receptor 3 Toll-Like , Humanos , Receptor 3 Toll-Like/metabolismo , Receptor 3 Toll-Like/genética , Quimiocina CXCL10/metabolismo , Quimiocina CXCL10/genética , Células Epiteliais/metabolismo , Células Epiteliais/efeitos dos fármacos , Brônquios/citologia , Brônquios/metabolismo , Poli I-C/farmacologia , Transdução de Sinais/efeitos dos fármacos , Linhagem Celular , Imunidade Inata , Interferon beta/metabolismo , Interferon beta/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas de Ligação a RNA , Proteínas Adaptadoras de Transdução de Sinal , Proteínas Reguladoras de Apoptose
2.
Amyloid ; 31(2): 105-115, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38343068

RESUMO

BACKGROUND: Dialysis-related amyloidosis (DRA) is a severe complication in end-stage kidney disease (ESKD) patients undergoing long-term dialysis treatment, characterized by the deposition of ß2-microglobulin-related amyloids (Aß2M amyloid). To inhibit DRA progression, hexadecyl-immobilized cellulose bead (HICB) columns are employed to adsorb circulating ß2-microglobulin (ß2M). However, it is possible that the HICB also adsorbs other molecules involved in amyloidogenesis. METHODS: We enrolled 14 ESKD patients using HICB columns for DRA treatment; proteins were extracted from HICBs following treatment and identified using liquid chromatography-linked mass spectrometry. We measured the removal rate of these proteins and examined the effect of those molecules on Aß2M amyloid fibril formation in vitro. RESULTS: We identified 200 proteins adsorbed by HICBs. Of these, 21 were also detected in the amyloid deposits in the carpal tunnels of patients with DRA. After passing through the HICB column and hemodialyzer, the serum levels of proteins such as ß2M, lysozyme, angiogenin, complement factor D and matrix Gla protein were reduced. These proteins acted in the Aß2M amyloid fibril formation. CONCLUSIONS: HICBs adsorbed diverse proteins in ESKD patients with DRA, including those detected in amyloid lesions. Direct hemoperfusion utilizing HICBs may play a role in acting Aß2M amyloidogenesis by reducing the amyloid-related proteins.


Assuntos
Amiloidose , Celulose , Falência Renal Crônica , Proteômica , Diálise Renal , Microglobulina beta-2 , Humanos , Amiloidose/metabolismo , Amiloidose/sangue , Amiloidose/terapia , Diálise Renal/efeitos adversos , Masculino , Feminino , Microglobulina beta-2/metabolismo , Microglobulina beta-2/sangue , Proteômica/métodos , Idoso , Celulose/química , Pessoa de Meia-Idade , Adsorção , Falência Renal Crônica/terapia , Falência Renal Crônica/metabolismo , Falência Renal Crônica/sangue , Espectrometria de Massas/métodos , Amiloide/metabolismo , Cromatografia Líquida
3.
Front Med (Lausanne) ; 9: 1036787, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36388880

RESUMO

Objective: Innate lymphoid cells (ILCs), including natural killer (NK) cells, ILC1, ILC2, lymphoid tissue-inducer (LTi) cells, and ILC3 cell, play a key role in various immune responses. Primary Sjögren's syndrome (pSS) is an autoimmune disease characterized by chronic inflammation of exocrine glands, such as the lacrimal and salivary glands (SGs). The role of NK cells among ILCs in the pathogenesis of pSS is still unclear. In this study, the characteristics and subsets of NK cells in the salivary gland (SG) tissue were analyzed using a murine model of pSS. Methods: Multiple phenotypes and cytotoxic signature of the SG NK cells in control and pSS model mice were evaluated by flow cytometric analysis. Intracellular expression of interferon-γ (IFN-γ) among T cells and NK cells from the SG tissues was compared by in vitro experiments. In addition, pathological analysis was performed using anti-asialo-GM1 (ASGM1) antibody (Ab)-injected pSS model mice. Results: The number of conventional NK (cNK) cells in the SG of pSS model mice significantly increased compared with that in control mice at 6 weeks of age. The production level of IFN-γ was significantly higher in SG NK cells than in SG T cells. The depletion of NK cells by ASGM1 Ab altered the ratio of tissue resident NK (rNK) cells to cNK cells, which inhibited the injury to SG cells with the recovery of saliva secretion in pSS model mice. Conclusion: The results indicate that SG cNK cells may enhance the autoreactive response in the target organ by upregulating of IFN-γ, whereas SG rNK cells protect target cells against T cell cytotoxicity. Therefore, the activation process and multiple functions of NK cells in the target organ could be helpful to develop potential markers for determining autoimmune disease activity and target molecules for incurable immune disorders.

4.
Am J Pathol ; 192(11): 1559-1572, 2022 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-35963465

RESUMO

The toxicologic effects of nanomaterials, such as carbon nanotubes (CNTs), on the immune system are understood well. However, the precise relationship between long-term exposure to CNTs and chronic inflammation remains unclear. In this study, a mouse model of chronic peritonitis was established using i.p. injection of multiwalled CNTs treated by the Taquann method with high dispersion efficiency. Chronic peritonitis with fibrosis was observed in Taquann-treated multiwalled CNT (T-CNT)-injected mice, but not in Taquann-treated titanium dioxide-injected mice. In vivo and in vitro experiments showed that matrix metalloproteinase-12 (MMP-12) of macrophages was up-regulated by T-CNT to enhance fibroblast activation and profibrotic molecule expression in fibroblasts. In addition, T-CNT-induced peritonitis reduced MMP-12 expression in Nfκb1-/- mice, suggesting that MMP-12-producing macrophages play a key role in chronic inflammation due to T-CNT exposure through NF-κB activation. The results of this study could be helpful in understanding the molecular toxicity of nanomaterial and chronic inflammation.

5.
Viruses ; 14(7)2022 07 04.
Artigo em Inglês | MEDLINE | ID: mdl-35891453

RESUMO

Sjögren's syndrome (SS) is an autoimmune disease especially targeting exocrine glands, such as the salivary and lacrimal glands. A radical therapy for SS based on its etiology has not been established because of the complex pathogenesis of the disease. Several studies have demonstrated a relationship between virus infection and SS pathogenesis. In particular, infection with the Epstein-Barr (EB) virus among others is a potent factor associated with the onset or development of SS. Specifically, virus infection in the target organs of SS triggers or promotes autoreactive responses involving the process of autoantigen formation, antigen-presenting function, or T-cell response. Our review of recent research highlights the crucial roles of virus infection in the pathogenesis of SS and discusses the critical association between virus infection and the etiology of autoimmunity in SS.


Assuntos
Síndrome de Sjogren , Viroses , Autoantígenos , Autoimunidade , Herpesvirus Humano 4 , Humanos , Síndrome de Sjogren/etiologia , Síndrome de Sjogren/patologia , Viroses/complicações
6.
Cell Death Dis ; 12(7): 698, 2021 07 13.
Artigo em Inglês | MEDLINE | ID: mdl-34257282

RESUMO

Sorafenib, a protein kinase inhibitor approved for the treatment of hepatocellular carcinoma and advanced renal cell carcinoma, has been repeatedly reported to induce ferroptosis by possibly involving inhibition of the cystine/glutamate antiporter, known as system xc-. Using a combination of well-defined genetically engineered tumor cell lines and canonical small molecule ferroptosis inhibitors, we now provide unequivocal evidence that sorafenib does not induce ferroptosis in a series of tumor cell lines unlike the cognate system xc- inhibitors sulfasalazine and erastin. We further show that only a subset of tumor cells dies by ferroptosis upon sulfasalazine and erastin treatment, implying that certain cell lines appear to be resistant to system xc- inhibition, while others undergo ferroptosis-independent cell death. From these findings, we conclude that sorafenib does not qualify as a bona fide ferroptosis inducer and that ferroptosis induced by system xc- inhibitors can only be achieved in a fraction of tumor cell lines despite robust expression of SLC7A11, the substrate-specific subunit of system xc-.


Assuntos
Antineoplásicos/farmacologia , Ferroptose/efeitos dos fármacos , Neoplasias/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Sorafenibe/farmacologia , Sistema y+ de Transporte de Aminoácidos/antagonistas & inibidores , Sistema y+ de Transporte de Aminoácidos/genética , Sistema y+ de Transporte de Aminoácidos/metabolismo , Animais , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos , Células HEK293 , Humanos , Camundongos , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , Piperazinas/farmacologia , Sulfassalazina/farmacologia
7.
Int J Mol Sci ; 22(6)2021 Mar 22.
Artigo em Inglês | MEDLINE | ID: mdl-33810172

RESUMO

The relationship between autoimmunity and changes in intestinal microbiota is not yet fully understood. In this study, the role of intestinal microbiota in the onset and progression of autoimmune lesions in non-obese diabetic (NOD) mice was evaluated by administering antibiotics to alter their intestinal microenvironment. Flow cytometric analysis of spleen cells showed that antibiotic administration did not change the proportion or number of T and B cells in NOD mice, and pathological analysis demonstrated that autoimmune lesions in the salivary glands and in the pancreas were also not affected by antibiotic administration. These results suggest that the onset and progression of autoimmunity may be independent of enteral microbiota changes. Our findings may be useful for determining the appropriate use of antibiotics in patients with autoimmune diseases who are prescribed drugs to maintain systemic immune function.


Assuntos
Antibacterianos/farmacologia , Doenças Autoimunes/etiologia , Autoimunidade , Suscetibilidade a Doenças , Animais , Antibacterianos/administração & dosagem , Antibacterianos/efeitos adversos , Doenças Autoimunes/tratamento farmacológico , Autoimunidade/efeitos dos fármacos , Citocinas/metabolismo , Modelos Animais de Doenças , Sistema Imunitário/efeitos dos fármacos , Sistema Imunitário/imunologia , Sistema Imunitário/metabolismo , Camundongos , Camundongos Endogâmicos NOD , Sialadenite/etiologia , Sialadenite/metabolismo , Sialadenite/patologia
8.
Ticks Tick Borne Dis ; 12(3): 101683, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33578257

RESUMO

We performed tick surveys in all regions (Kaetsu, Chuetsu, Joetsu, and Sado) of the Niigata prefecture, Japan. A total of 105 field surveys were done from 2016 to 2018 in 41 sites, from north to south, in the prefecture. All 4806 ticks collected were identified and classified by species, sex, and developmental stage. Twelve species were recorded: Dermacentor taiwanensis, Haemaphysalis flava, Haemaphysalis hystricis, Haemaphysalis japonica, Haemaphysalis longicornis, Haemaphysalis megaspinosa, Ixodes ovatus, Ixodes nipponensis, Ixodes persulcatus, Ixodes monospinosus, Ixodes columnae, and Ixodes turdus. The major tick species in Niigata prefecture were H. flava, H. longicornis, and I. ovatus and they comprised 93.4% of all samples. These three species have one generation per year. Climatic and anthropogenic factors may be involved in the substantial change of the endemic species composition from a previous tick survey (1959) in the Niigata prefecture. These factors include increasing temperatures, introduction of new hosts such as the wild boar, highway construction, and a rural exodus facilitating animal migration and reproduction. Tick hosts suitable for the transmission of Japanese spotted fever, Lyme borreliosis, and SFTS occur in Niigata prefecture. Heightened awareness of these three tick-borne diseases is needed for preparation and disease prevention.


Assuntos
Distribuição Animal , Biodiversidade , Ixodidae/fisiologia , Animais , Feminino , Ixodidae/crescimento & desenvolvimento , Japão , Larva/crescimento & desenvolvimento , Larva/fisiologia , Masculino , Ninfa/crescimento & desenvolvimento , Ninfa/fisiologia , Estações do Ano
9.
J Am Chem Soc ; 142(43): 18522-18531, 2020 10 28.
Artigo em Inglês | MEDLINE | ID: mdl-33047956

RESUMO

Expanding the repertoire of electrophiles with unique reactivity features would facilitate the development of covalent inhibitors with desirable reactivity profiles. We herein introduce bicyclo[1.1.0]butane (BCB) carboxylic amide as a new class of thiol-reactive electrophiles for selective and irreversible inhibition of targeted proteins. We first streamlined the synthetic routes to generate a variety of BCB amides. The strain-driven nucleophilic addition to BCB amides proceeded chemoselectively with cysteine thiols under neutral aqueous conditions, the rate of which was significantly slower than that of acrylamide. This reactivity profile of BCB amide was successfully exploited to develop covalent ligands targeting Bruton's tyrosine kinase (BTK). By tuning BCB amide reactivity and optimizing its disposition on the ligand, we obtained a selective covalent inhibitor of BTK. The in-gel activity-based protein profiling and mass spectrometry-based chemical proteomics revealed that the selected BCB amide had a higher target selectivity for BTK in human cells than did a Michael acceptor probe. Further chemical proteomic study revealed that BTK probes bearing different classes of electrophiles exhibited distinct off-target profiles. This result suggests that incorporation of BCB amide as a cysteine-directed electrophile could expand the capability to develop covalent inhibitors with the desired proteome reactivity profile.


Assuntos
Tirosina Quinase da Agamaglobulinemia/antagonistas & inibidores , Amidas/química , Compostos Bicíclicos com Pontes/química , Cisteína/química , Tirosina Quinase da Agamaglobulinemia/metabolismo , Linhagem Celular , Ciclobutanos/química , Humanos , Ligantes , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/metabolismo
10.
Int J Cancer ; 147(11): 3224-3235, 2020 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-32818320

RESUMO

The cystine/glutamate antiporter, system xc- , is essential for the efficient uptake of cystine into cells. Interest in the mechanisms of system xc- function soared with the recognition that system xc- presents the most upstream node of ferroptosis, a recently described form of regulated necrosis relevant for degenerative diseases and cancer. Since targeting system xc- hold the great potential to efficiently combat tumor growth and metastasis of certain tumors, we disrupted the substrate-specific subunit of system xc- , xCT (SLC7A11) in the highly metastatic mouse B16F10 melanoma cell line and assessed the impact on tumor growth and metastasis. Subcutaneous injection of tumor cells into the syngeneic B16F10 mouse melanoma model uncovered a marked decrease in the tumor-forming ability and growth of KO cells compared to control cell lines. Strikingly, the metastatic potential of KO cells was markedly reduced as shown in several in vivo models of experimental and spontaneous metastasis. Accordingly, survival rates of KO tumor-bearing mice were significantly prolonged in contrast to those transplanted with control cells. Analyzing the in vitro ability of KO and control B16F10 cells in terms of endothelial cell adhesion and spheroid formation revealed that xCT expression indeed plays an important role during metastasis. Hence, system xc- emerges to be essential for tumor metastasis in mice, thus qualifying as a highly attractive anticancer drug target, particularly in light of its dispensable role for normal life in mice.


Assuntos
Sistema y+ de Transporte de Aminoácidos/genética , Técnicas de Inativação de Genes/métodos , Melanoma/patologia , Animais , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Melanoma/genética , Camundongos , Metástase Neoplásica , Transplante de Neoplasias , Taxa de Sobrevida
11.
ACS Med Chem Lett ; 11(6): 1137-1144, 2020 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-32550993

RESUMO

Covalent modification of disease-associated proteins with small molecules is a powerful approach for achieving an increased and sustained pharmacological effect. To reduce the potential risk of nonselective covalent modification, molecular design of covalent inhibitors is critically important. We report herein the development of a targeted covalent inhibitor for mutated epidermal growth factor receptor (EGFR) (L858R/T790M) using α-chlorofluoroacetamide (CFA) as the reactive group. The chemically tuned weak reactivity of CFA was suitable for the design of third-generation EGFR inhibitors that possess the pyrimidine scaffold. The structure-activity relationship study revealed that CFA inhibitor 18 (NSP-037) possessed higher inhibition selectivity to the mutated EGFR over wild-type EGFR when compared to clinically approved osimertinib. Mass-based chemical proteomics analyses further revealed that 18 displayed high covalent modification selectivity for the mutated EGFR in living cells. These findings highlight the utility of CFA as a warhead of targeted covalent inhibitors and the potential application of the CFA-pyrimidines for treatment of non-small-cell lung cancer.

12.
Planta ; 251(4): 78, 2020 Mar 10.
Artigo em Inglês | MEDLINE | ID: mdl-32157441

RESUMO

MAIN CONCLUSION: A hypothesis that squalene cyclase genes are widely distributed throughout ferns was proposed. We successfully isolated a squalene cyclase pseudogene from a fern from which no triterpene hydrocarbons were detected Ferns are the most primitive vascular plants, with their locations ranging from tropical to cold temperate regions and from lowland to alpine zones. The triterpene hydrocarbons and their derivatives are characteristic fern metabolites, and are also chemophenetic markers. Recently, our biosynthetic study into fern squalene cyclases (SCs), the enzymes responsible for triterpene synthesis, gave an unexpected inconsistency between genotype (enzyme function) and chemotype (triterpene profile). This finding prompted us to propose a hypothesis that SC genes are widely distributed throughout ferns and lycophytes whether or not they produce triterpene hydrocarbons. To test this hypothesis, we employed a multifaceted approach based on phytochemical, biochemical, and phylogenetic analyses. As anticipated, we successfully isolated two SC pseudogenes from a fern from in which no or only one triterpene hydrocarbon was detected. Subsequent mutagenesis experiments resulted in the functional conversion of these pseudogenes into active SC genes. Given an auxiliary hypothesis regarding the inherent limit of the degenerate polymerase chain reaction (PCR) method, the overall dataset supported our hypothesis, although correction was required with respect to plant coverage. Not only did the corrected hypothesis outline the distribution of SC genes throughout ferns, it provided insight into the molecular basis of the triterpene-based chemophenetics in ferns, which is also discussed.


Assuntos
Gleiquênias/metabolismo , Triterpenos/metabolismo , Clonagem Molecular , Evolução Molecular , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Liases/genética , Liases/metabolismo , Espectroscopia de Ressonância Magnética , Biologia Molecular , Filogenia , Pichia/genética , Alinhamento de Sequência
13.
J Biol Chem ; 295(12): 4014-4023, 2020 03 20.
Artigo em Inglês | MEDLINE | ID: mdl-32041779

RESUMO

Inorganic polyphosphate (polyP) is a linear polymer of orthophosphate units that are linked by phosphoanhydride bonds and is involved in various pathophysiological processes. However, the role of polyP in immune cell dysfunction is not well-understood. In this study, using several biochemical and cell biology approaches, including cytokine assays, immunofluorescence microscopy, receptor-binding assays with quartz crystal microbalance, and dynamic light scanning, we investigated the effect of polyP on in vitro lipopolysaccharide (LPS)-induced macrophage inflammatory response. PolyP up-regulated LPS-induced production of the inflammatory cytokines, such as tumor necrosis factor α, interleukin-1ß, and interleukin-6, in macrophages, and the effect was polyP dose- and chain length-dependent. However, orthophosphate did not exhibit this effect. PolyP enhanced the LPS-induced intracellular macrophage inflammatory signals. Affinity analysis revealed that polyP interacts with LPS, inducing formation of small micelles, and the polyP-LPS complex enhanced the binding affinity of LPS to Toll-like receptor 4 (TLR4) on macrophages. These results suggest that inorganic polyP plays a critical role in promoting inflammatory response by enhancing the interaction between LPS and TLR4 in macrophages.


Assuntos
Citocinas/metabolismo , Fosfatos/farmacologia , Regulação para Cima/efeitos dos fármacos , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Citocinas/genética , Humanos , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Interleucina-6/genética , Interleucina-6/metabolismo , Lipopolissacarídeos/metabolismo , Lipopolissacarídeos/farmacologia , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , NF-kappa B/antagonistas & inibidores , NF-kappa B/metabolismo , Nitrilas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Sulfonamidas/farmacologia , Sulfonas/farmacologia , Receptor 4 Toll-Like/agonistas , Receptor 4 Toll-Like/metabolismo , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo
14.
Nature ; 575(7784): 693-698, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31634899

RESUMO

Ferroptosis is an iron-dependent form of necrotic cell death marked by oxidative damage to phospholipids1,2. To date, ferroptosis has been thought to be controlled only by the phospholipid hydroperoxide-reducing enzyme glutathione peroxidase 4 (GPX4)3,4 and radical-trapping antioxidants5,6. However, elucidation of the factors that underlie the sensitivity of a given cell type to ferroptosis7 is crucial to understand the pathophysiological role of ferroptosis and how it may be exploited for the treatment of cancer. Although metabolic constraints8 and phospholipid composition9,10 contribute to ferroptosis sensitivity, no cell-autonomous mechanisms have been identified that account for the resistance of cells to ferroptosis. Here we used an expression cloning approach to identify genes in human cancer cells that are able to complement the loss of GPX4. We found that the flavoprotein apoptosis-inducing factor mitochondria-associated 2 (AIFM2) is a previously unrecognized anti-ferroptotic gene. AIFM2, which we renamed ferroptosis suppressor protein 1 (FSP1) and which was initially described as a pro-apoptotic gene11, confers protection against ferroptosis elicited by GPX4 deletion. We further demonstrate that the suppression of ferroptosis by FSP1 is mediated by ubiquinone (also known as coenzyme Q10, CoQ10): the reduced form, ubiquinol, traps lipid peroxyl radicals that mediate lipid peroxidation, whereas FSP1 catalyses the regeneration of CoQ10 using NAD(P)H. Pharmacological targeting of FSP1 strongly synergizes with GPX4 inhibitors to trigger ferroptosis in a number of cancer entities. In conclusion, the FSP1-CoQ10-NAD(P)H pathway exists as a stand-alone parallel system, which co-operates with GPX4 and glutathione to suppress phospholipid peroxidation and ferroptosis.


Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Ferroptose/genética , Glutationa/metabolismo , Proteínas Mitocondriais/metabolismo , Animais , Proteínas Reguladoras de Apoptose/genética , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Técnicas de Inativação de Genes , Humanos , Peroxidação de Lipídeos/genética , Camundongos , Proteínas Mitocondriais/genética , Ubiquinona/análogos & derivados , Ubiquinona/metabolismo
15.
Am J Pathol ; 189(12): 2414-2427, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31539517

RESUMO

Follicular helper T (Tfh) cells contribute to various immune responses as well as to the pathogenesis of several immune diseases. However, the precise mechanism underlying the onset or development of autoimmunity via Tfh cells remains unclear. Herein, the detailed relationship between autoimmune disease and Tfh cells was analyzed using a murine model for Sjögren syndrome (SS) wherein the mice underwent neonatal thymectomy. Germinal center (GC) development was promoted in this SS model along with an increase of Tfh cells and GC B cells. The severity of the autoimmune lesions was correlated with the number of Tfh cells detected in the spleen of the SS model mice. In addition, treatment with an anti-CD20 monoclonal antibody effectively suppressed the autoimmune lesions with a reduction of Tfh cells and GC B cells. Comprehensive gene analysis revealed that several genes associated with Tfh cell differentiation, including achaete-scute homologue 2 (Ascl2), were up-regulated in peripheral CD25- CD4+ T cells in SS model mice compared with those in control mice. Moreover, an experiment using CD4CreBcl6fl/fl mice that received neonatal thymectomy treatment demonstrated that Ascl2 contributes to the Tfh cell differentiation associated with autoimmunity during the early stages, independent of Bcl6. In conclusion, our results indicate that abnormal Tfh cell differentiation via Ascl2 regulation might contribute to the pathogenesis of autoimmunity.


Assuntos
Autoimunidade/imunologia , Linfócitos B/imunologia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Modelos Animais de Doenças , Centro Germinativo/imunologia , Síndrome de Sjogren/imunologia , Linfócitos T Auxiliares-Indutores/imunologia , Animais , Autoanticorpos/imunologia , Autoanticorpos/metabolismo , Linfócitos B/metabolismo , Linfócitos B/patologia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Diferenciação Celular , Citocinas/imunologia , Citocinas/metabolismo , Feminino , Centro Germinativo/metabolismo , Centro Germinativo/patologia , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Proto-Oncogênicas c-bcl-6/genética , Proteínas Proto-Oncogênicas c-bcl-6/metabolismo , Síndrome de Sjogren/metabolismo , Síndrome de Sjogren/patologia , Linfócitos T Auxiliares-Indutores/metabolismo , Linfócitos T Auxiliares-Indutores/patologia
16.
J Oral Biosci ; 61(3): 179-182, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31400544

RESUMO

OBJECTIVES: Sicca syndrome is characterized by dry mouth and eyes and results in a reduction of the patient's quality of life. Various natural plants, including certain herbs, have long been employed to alleviate such symptoms. Rooibos grown in South Africa is one of the potent herbal plants used for the treating dry mouth. However, the precise mechanism of action by which rooibos alleviates symptoms of dryness remains unclear. METHODS: The in vivo effects of rooibos extract (RE), which comprises eriodictyol-6-C-glucoside, on the secretory function of saliva and tears were analyzed after intraoral RE administration using wild-type C57BL/6 (B6) mice. In addition, the mechanisms of RE were investigated after administration of a muscarinic acetylcholine receptor 3 (M3R) antagonist. RESULTS: Tear and saliva volumes in mice increased significantly and in a dose-dependent manner following intraoral RE administration compared to those in mice in the control group administered H2O. An experiment performed using darifenacin administration revealed that the effects of RE on secretory function were exerted via M3R. CONCLUSION: These results suggest that RE administration is an effective treatment for symptoms of dryness and may be used in clinical settings against sicca syndrome.


Assuntos
Aspalathus , Animais , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Extratos Vegetais , Qualidade de Vida , Receptores Muscarínicos , Saliva , África do Sul
17.
Blood Purif ; 48(3): 215-222, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31055586

RESUMO

BACKGROUND/AIMS: Accumulation of protein-bound uremic toxins (PBUTs) is associated with mortality due to various systemic disorders in patients with chronic kidney disease (CKD), especially in those undergoing dialysis treatment. The clinical outcomes of such patients could be improved by removing sufficient amounts of PBUTs; however, conventional dialysis lacks this ability. We examined the efficacy of activated carbon in adsorbing circulating PBUTs through direct hemoperfusion (DHP) in vitro. METHODS: An in vitro blood circulating system was constructed with 8.5 mL blood circulating around a column containing activated carbon (50, 100, or 200 mg). Bovine blood containing a kind of PBUT (at the same concentration as that found in the blood of dialysis patients) and blood from hemodialysis patients (n = 8) were used. After circulation for the designated amount of time, sera were collected and the levels of PBUTs, including indoxyl sulfate (IS), p-cresyl sulfate, indole acetic acid (IAA), phenyl sulfate, and hippuric acid, were analyzed with mass spectrometry. RESULTS: Activated carbon decreased the PBUT level in bovine blood in a dose-dependent manner (e.g., reduction rate of IS: 67.9 ± 3.8, 83.3 ± 1.9, and 94.5 ± 1.1% after 60-min circulation in columns containing 50, 100, and 200 mg activated carbon respectively). IS, PCS, and IAA were dramatically adsorbed by activated carbon from the blood of patients undergoing hemodialysis (pre vs. post 240-min reaction: IS 2.835 ± 0.876 vs. 0.455 ± 0.108 mg/dL [p < 0.01], PCS 3.208 ± 2.876 vs. 0.768 ± 0.632 mg/dL [p < 0.01], IAA 0.082 ± 0.045 vs. 0.016 ± 0.005 mg/dL [p < 0.01]). CONCLUSION: Activated carbon effectively adsorbed blood PBUTs in vitro. DHP with activated carbon could be a promising strategy for removing circulating PBUTs from the blood of patients with CKD.


Assuntos
Hemoperfusão/métodos , Insuficiência Renal Crônica/terapia , Toxinas Biológicas/sangue , Uremia/terapia , Adsorção , Animais , Bovinos , Carvão Vegetal/química , Feminino , Humanos , Masculino , Espectrometria de Massas , Ligação Proteica , Toxinas Biológicas/isolamento & purificação
18.
Nat Chem Biol ; 15(3): 250-258, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30643284

RESUMO

Irreversible inhibition of disease-associated proteins with small molecules is a powerful approach for achieving increased and sustained pharmacological potency. Here, we introduce α-chlorofluoroacetamide (CFA) as a novel warhead of targeted covalent inhibitor (TCI). Despite weak intrinsic reactivity, CFA-appended quinazoline showed high reactivity toward Cys797 of epidermal growth factor receptor (EGFR). In cells, CFA-quinazoline showed higher target specificity for EGFR than the corresponding Michael acceptors in a wide concentration range (0.1-10 µM). The cysteine adduct of the CFA derivative was susceptible to hydrolysis and reversibly yielded intact thiol but was stable in solvent-sequestered ATP-binding pocket of EGFR. This environment-dependent hydrolysis can potentially reduce off-target protein modification by CFA-based drugs. Oral administration of CFA quinazoline NS-062 significantly suppressed tumor growth in a mouse xenograft model. Further, CFA-appended pyrazolopyrimidine irreversibly inhibited Bruton's tyrosine kinase with higher target specificity. These results demonstrate the utility of CFA as a new class warheads for TCI.


Assuntos
Acetamidas/síntese química , Cisteína/metabolismo , Quinazolinas/síntese química , Acetamidas/química , Acetamidas/farmacologia , Animais , Antineoplásicos , Linhagem Celular , Receptores ErbB , Humanos , Camundongos , Camundongos Nus , Neoplasias , Fosfotransferases/fisiologia , Inibidores de Proteínas Quinases/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Pirimidinas/antagonistas & inibidores , Quinazolinas/química , Relação Estrutura-Atividade , Ensaios Antitumorais Modelo de Xenoenxerto
19.
J Gastroenterol ; 54(6): 521-529, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30519747

RESUMO

BACKGROUND: Transabdominal ultrasonography (US) has been reported to be a useful tool for evaluating ulcerative colitis (UC) although with less well-established data than for Crohn's disease. This prospective multicenter study aimed to establish the usefulness of US compared with colonoscopy (CS) for assessing disease extent and activity of UC. METHODS: Altogether, 173 patients with UC were prospectively enrolled, among whom 156 were eligible for this study. All patients underwent US and CS within 2 days at five facilities. We divided the colon into six segments and examined each segment and the rectum using US and CS. US severity was graded 1-4 regarding bowel wall thickness, stratification, and ulceration. CS severity was also graded 1-4 according to Matts' endoscopic classification. Concordance between US and CS grades for all colonic segments was analyzed using kappa statistics. US and CS findings were also compared with the clinical disease activity index (CAI) and histological grade using Spearman's correlation coefficient. RESULTS: There was moderate concordance between US and CS grades in all colonic segments (weighted κ = 0.55, p < 0.001). Concordance was rated moderate for each colonic segment but only slight for the rectum. The US grade was significantly correlated with the CAI score (r = 0.40, p < 0.001) and histological grade (r = 0.35, p < 0.001). CONCLUSIONS: This prospective multicenter study showed moderate concordance between US and CS for assessing the disease activity of UC. Hence, US may be used more generally for evaluating UC in daily clinical practice.


Assuntos
Colite Ulcerativa/diagnóstico por imagem , Colonoscopia/métodos , Ultrassonografia/métodos , Adulto , Colite Ulcerativa/diagnóstico , Colite Ulcerativa/fisiopatologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Índice de Gravidade de Doença
20.
Nitric Oxide ; 78: 32-40, 2018 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-29792932

RESUMO

The amino acid transport system xc- is important for maintaining intracellular glutathione levels and extracellular redox balance. The main component of system xc-, xCT, is strongly induced by various stimuli, including oxidative stress and bacterial lipopolysaccharides (LPS) in macrophages. In the present study, we investigated the production of nitric oxide by LPS-stimulated mouse peritoneal macrophages isolated from both xCT-deficient and wild-type mice. After culturing macrophages in the presence of LPS for 24-48 h, nitrite levels in the medium of xCT-deficient macrophages were significantly decreased compared to that of wild-type cells. However, the transport activity of arginine, a precursor of nitric oxide, and the expression of nitric oxide synthase 2 in xCT-deficient macrophages were similar to those of wild-type cells. When wild-type macrophages were cultured in the medium that contained no cystine, nitric oxide production was decreased to the level similar to that of the xCT-deficient macrophages. When xCT-deficient macrophages were cultured with 2-mercaptoethanol, intracellular cysteine levels were increased and nitrite accumulation in the medium was significantly increased. On the other hand, when these cells were cultured with buthionine sulfoximine, an inhibitor of glutathione synthesis, nitrite accumulation in the medium was essentially unchanged, although intracellular glutathione levels were very low. Reactive oxygen species levels in xCT-deficient macrophages were higher than those of wild-type cells, and treatment with LPS caused an increase in oxidative stress in both cells. These results suggest that intracellular cysteine supplied by xCT contributes to nitric oxide production and the reduction of oxidative stress in macrophages.


Assuntos
Sistema y+ de Transporte de Aminoácidos/deficiência , Macrófagos Peritoneais/metabolismo , Óxido Nítrico/metabolismo , Sistema y+ de Transporte de Aminoácidos/genética , Animais , Cisteína/metabolismo , Cistina/metabolismo , Glutationa/metabolismo , Mercaptoetanol/farmacologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Estresse Oxidativo/fisiologia , Espécies Reativas de Oxigênio/metabolismo
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